Type I interferons (IFNs) are well-characterized cytokines that inhibit a wide range of viruses. The cellular proteins that mediate the antiviral functions of IFN are derived from interferon-stimulated genes (ISGs), and are potentially exploitable for the development of pan-tropic antiviral drugs. Unfortunately, very little is known about which ISGs are antiviral, their specificity is largely undetermined, and their mechanisms of action remain elusive. We propose to address this gap in scientific knowledge by conducting a series of large-scale screens to determine the ISG profile of diverse viruses. We have cloned over 380 commonly upregulated ISGs into a lentiviral vector and optimized conditions to correlate the expression of these genes with viral replication using a two-color flow cytometric assay. The ISG library will be used to probe ISG antiviral function against an array of viruses form diverse families, with a special emphasis on hepatitis C virus (HCV) and influenza virus A (FluA). Each of these viruses has a significant impact on global health, with pathologies ranging from hepatocellular carcinoma to severe respiratory disease, and potential outcomes spanning life-long chronic infection to acute pandemic events. Comparison of ISG profiles from across viral species, genera, and families will begin to reveal the range of ISG specificities. Screens of select viruses under different cellular conditions will address the depth of ISG function. Validation and mechanistic dissection of the most interesting hits will clarify the roles of ISGs in combating infection, and may yield new strategies for translating naturally occurring broad-spectrum antiviral activities into clinically useful compounds.